PROJECT SUMMARY/ABSTRACT Norovirus is the leading cause of epidemic gastroenteritis worldwide, but lacks an effective vaccine or antiviral treatment. After infection, virus can be shed chronically for weeks to months, potentially contributing to future outbreaks. A small animal model for persistent norovirus infection revealed that the commensal bacterial microbiome enhances norovirus infection. Interferon-lambda (IFN-?), an innate immune cytokine, plays an important role in this process via an unclear mechanism. IFN-? stimulates antiviral signaling on intestinal epithelial cells, the same cells infected by persistent norovirus in vivo, and can prevent or cure infection. Commensal bacteria may therefore promote norovirus infection by preventing host IFN-? responses to virus. Recent data also revealed that altered microbial communities in immunocompromised mice are associated with excessive IFN-? and norovirus resistance. A unifying hypothesis for these findings is that specific bacteria diminish or enhance the capacity of intestinal epithelial cells to generate IFN-? to regulate norovirus. Identification of specific commensal bacteria that promote norovirus is critical to understanding in vivo viral regulation. Dilutional fecal transplants and colonization experiments revealed a promising bacterial candidate; comparison of this candidate to a related species that does not promote norovirus will highlight bacterial phenotypic characteristics determining viral infection. The effects of this bacteria on specific metabolites, as well as assessment of its localization in proximity to norovirus-infected cells in vivo, will be explored. This proposal will also evaluate how this candidate bacteria regulates norovirus-permissive tuft cells. Preliminary data indicates that bacterial products prevent IFN-? induction by norovirus in vitro. Reporter mice will be used to assay in vivo regulation of interferon signaling and viral infection by bacteria. The mechanisms by which bacteria alter transcriptional responses to virus will also be interrogated in a novel in vitro intestinal epithelial cell model. Finally, a fecal factor in immunocompromised mice confers norovirus resistance when transferred, and correlates with excessive IFN-? and altered intestinal bacteria. The role of IFN-? in this viral resistance will be tested using mouse lines lacking the IFN-?-receptor, and the transferable factor will be identified by treating transferred material and testing candidate isolates. Because the immunocompromised mice have broad adaptive immune defects, different adaptive immune factors will be tested for their regulation of the microbiome and the transferable factor. Completion of this proposal will provide mechanistic understanding into the regulation of intestinal innate immune signaling and norovirus infection by commensal bacteria. These studies will reveal bacteria with modulatory effects on the intestinal epithelium to adapt for probiotic strategies to combat enteric viruses.